1. Field of the Invention
The invention pertains to a multi layered cell harvester formed from a plurality of flat substantially rectangular layers having channels and passages that interconnect a paired set of wash and sample removal needles for automatically removing and harvesting cells from a plurality of test tubes or cells containing small amounts of liquid arranged in standard 96, 48 or other test tube arrangement of arrays. More particularly the invention pertains to a novel multi layered cell harvester which automatically removes cells from standard test tube racks using a plurality of sample removal needles then washes each of the test tubes by adding a controlled volume of wash fluid to each of the test tubes through a plurality of wash or reagent addition needles and then removes the wash fluid or reagent from each of the test tubes through the sample removal needles. The invention is particularly adapted to applications where the precise volumetric addition of reagents or wash fluids must be critically controlled in an array of test tubes or cells containing small volumes of fluid.
The invention provides for the precise and equivalent distribution of reagents or wash fluids to all test tubes in an array by equally adding fluid to each test tube irrespective of its position in the array by volumetrically maintaining equivalence between each of the test tube in the array. The volumetric equivalence between the test tubes in the array is provided by maintaining an equivalent length of channels from a reservoir to the tip of each wash needle, or increasing the diameter of some of the channels or by a combination of length and diameter of the channel to provide an equal volumetric addition of fluid to each test tube irrespective of its position in the test tube rack or array.
The invention is particularly suitable to applications where precise amounts of wash fluids or reagents must be precisely controlled or where small volumes of sample containers are disposed in an array or test tube rack which would overflow if a precise control of the wash fluid or added reagents were not controlled. The invention is therefore particularly advantageously adapted to a large number of small test tubes in a standard 96 or 48 test tube rack. The standard 96 or 48 test tube rack for harvesting of cells measures about 5 inches (12.8 cm) by about 31/4inches (8.3 cm). The standard test tube array used in the standard 96 test tube rack is arranged in 8 rows each containing 12 small test tubes which measure about 2 inches long (5.1 cm) having a diameter of about 1/4 of an inch (6 mm) with a center distance of about 3/8th of an inch (1 cm) in which cells are placed in various fluids or mother liquids to evaluate their response to a variety of drugs and pharmaceutical preparations that in some cases include radioactive tagging agents.
The standard procedure is to remove the contents of each of the small test tubes and filter the contents of each of the small test tubes onto a sheet of filter paper having 96 separate circular areas of about 1/2 of an inch in diameter (1.3 cm). The samples from the 96 test tubes from the standard array are disposed in 96 separate 1/2inch (1.3) circular areas on a filter paper measuring approximately 93/4 (24.5 cm) by 63/4inch (17.3 cm). The sheet of filter paper containing the 96 separate 1/2 inch circular areas after filtration and washing the test tube samples onto the paper is subsequently cut and cultivated, placed in scintillation readers or otherwise treated to determine the efficacy of the various pharmaceutical preparations.
In operation it is important that cross contamination of the samples is avoided and separation of the samples is maintained as well as in some cases saving the mother liquid and wash reagents. As is known to those skilled in the art, the manual steps of removing, filtering and maintaining the segregation of the filtered sample and the washing of the sample out of the test tubes is a laborious and expensive process. The process can not only be expensive but dangerous to employees since some of the materials are hazardous and the possibility of contamination of the laboratory personnel is increased by the number of manipulative steps required in washing samples out of the test tubes and cleaning of the cell harvesting apparatus after each operation.
The present invention not only reduces these steps by automatically filtering and washing cell cultures from an array of test tubes but also effectively and automatically maintains the volume of wash fluids or reagents to each of the test tubes to prevent them from overflowing while maintaining the segregation of the filtered samples from the test tube onto the 1/2 inch (1.3 cm) areas on the filter paper. The apparatus of the invention is readily adaptable to robotic operation and eliminates much of the tubing and external apparatus that are conventionally employed in the laboratory that can increase the risk of contamination of employees when the hoses or apparatus require maintenance or cleaning The apparatus of the invention can be easily sterilized chemically or by radiation or can be sterilized by heating in an autoclave when the multi-layered cell harvester is made out of metal.
2. Description of the Prior Art
The prior art includes a variety of available filtration apparatus and devices for harvesting cell cultures. The available devices have expedited and automated a lot of the laboratory procedures involved in removing samples from large volume laboratory test tubes and racks but have still required a number of operative steps by the laboratory technician to remove the samples and if necessary save the wash reagents and mother liquid while making certain to completely remove and wash out the contents of the test tube which can be critical to determine quantitative results in binding studies. Some of the prior art devices have used vacuum sources which required turning over the test tubes, separate washing steps or the utilization of special handling equipment to change from large volume test tubes to the small volume test tubes used in standard test tube racks to provide a full 1/2 inch (1.3 cm) filtering area for the sample. Small volume test tubes are generally preferred in biological studies in view of costs of reagents and availability of compounds which then should be filtered on a 1/2 inch (1.3 cm) filter area that can be cut from the filter paper and subsequently used in cell scintillation studies, read on scintillation readers or otherwise processed.
The standard and preferred arrangement for the harvesting of cells employs a 96 or 48 test tube rack with test tubes about 2 inches long (5.1 cm) with a 1/4 inch (6 mm) diameter opening arranged in 8 rows. In some cases these test tubes are connected to one another in columns of twelve test tubes to provide a column of twelve test tubes measuring about 4 inches long (10.6 cm). The length of each test tube is about 2 inches long (5 cm), with a diameter of about 1/4 of an inch (6 mm) with the center distance between each of the tubes being about 3/8ths of an inch (1 cm) in each row. The rows are generally separated from one another by about 3/8th of an inch (1 cm) to form a substantially rectangular 96 test tube array. Sometimes only 4 rows of these 12 column test tubes are used in the 96 test tube rack or a special 48 test tube rack can be employed. In all such arrangements the samples must be removed and filtered and the test tubes precisely washed with an equal volume of wash fluid or reagent.
The problem is to remove the samples from each of the test tubes in the array, provide an equal volume of wash fluid or reagent to each of the test tubes irrespective of its position in the array to assure all of the sample is removed from the test tube and filter the contents of each of the test tube onto a circular 1/2 inch (1.3 cm) area. These circular areas containing the filtered culture are subsequently cut out and placed in a circular vials of greater than 1/2 inch diameter (1.3 cm) for further binding studies, read on scintillation counters or further processed according to the required studies.
Many of the available prior art devices require numerous operational steps for the removal of samples from the small tubes before filtering the contents of the test tubes on the 1/2 inch diameter (1.3 cm) areas of the filter paper as employed in pharmaceutical studies. A number of devices are available in the prior art which similarly provide a means for removing cells from test tubes but do not use a full 1/2 inch diameter (1.3 cm) filtering area but instead use a filtering area of about 1/4 inch in diameter (6 mm). These nonstandard systems have developed as a result of the difficulty in removing, washing and filtering samples from a standard 96 rack array having test tubes of the size heretofore described.
Many other prior art systems have utilized larger test tubes and racks in an effort to simplify the filtering and handling of cell cultures at the expense of utilizing greater quantities of reagents and pharmaceutical compounds that many times are available in limited quantities and made specially for cell binding studies. The trade off in the prior art has generally been in favor of the utilization of small test tubes and quantities of material at the expense of the extra procedural and manipulative steps required to effectively remove, wash and filter the samples. Large test tubes have not been favored because even through they are more easily handled and washed, they require greater amounts of reagents, compositions and tissues in order to complete the binding studies.
The present invention is applicable to both large and small test tubes but is particularly amenable to small test tube since it volumetrically controls the amount of wash fluid introduced into each test tube irrespective of its position in the test tube rack. This precise volumetric control prevents one or more tubes at the center of the test tube rack from overflowing with wash fluid or added reagent while test tubes at the periphery of the rack are only partially filled. The invention provides a critical control on the amount and even distribution of wash fluid to all of the test tubes in the rack irrespective of their position in the test tube rack.
Representative of prior art showing the utilization of a standard 96 test tube rack and the dispensing of fluid into the rack is Lancaster U.S. Pat. No. 3,650,306 which describes the problem of providing an automatic sampling and dispensing apparatus capable of precisely and simultaneously withdrawing into a plurality of needles or pipettes a predetermined reproducible microquantity of liquid from a liquid source and delivering the same to a plurality of corresponding wells in a micro-titration plate. This patent pertains to a laboratory dispensing apparatus and does not pertain to a system for simultaneously withdrawing the samples from the test tubes, filtering the samples on 1/2 inch (1.3 cm) diameter area on a sheet of filter paper and subsequently washing the test tube and the probes or needles for withdrawing the test tube samples. This patent demonstrates the problem of accurately dispensing a precise microquantity of liquid in each of the test tubes in the array but does not teach or suggest the more important problem of removing, filtering and subsequently refilling and washing each of the test tubes.
Some of the prior art pertains to a system for washing and aspirating wash fluid from the sample. Representative of such prior art is Dodge, et al. U.S. Pat. 3,949,771 which employs a single probe for removing and providing solution to a row of four vials. Dodge, et al U.S. Pat. No. 3,949,771 provides for the removal and washing of fluids, it does not filter or employ multi-layers to replace tubing or provide a system for simultaneously removing, filtering and washing the test tubes.
Many of the prior art systems employ large test tubes such as Leder et al. U.S. Pat. No. 3,319,792 and devices for removing and filtering samples. Shepel U.S. Pat. No. 4,317,726 similarly provides a filter assembly by forming a sandwich between a cover plate and a base plate but does not automatically remove the sample from the test tube and wash the test tube.
Weinstein, et al. U.S. Pat. No. 4,245,042 like many of the other prior art devices does not employ a standard 96 test tube rack but instead employs a standard cultivation plate having two consecutive rows of twelve conventional size wells. Weinstein, et al '042 provides inlet tubes for removing and filtering samples from the test tube and outlet tubelets for washing the conventional sized wells. Weinstein, et al '042, however, most importantly, does not provide a system for volumetrically controlling the amount of wash fluid introduced to each test tube irrespective of its position in the array and does not utilize a standard 96 rack or the eight rows of twelve test tubes which require a far more precise washing and evacuation technique to remove and filter samples on a 1/2 inch in diameter (1.3 cm) filter area arranged on a sheet of filter paper having a size of about 93/4 inch (25 cm) by 63/4 (17.3 cm). Weinstein, et al U.S. Pat. No. 4,245,042, since it only pertains to two rows of twelve conventional size wells and a standard cultivation plate did not have the critical control problem of supplying a precise volume of wash fluid to each sample which requires the utilization of wash channels to provide a constant volume of water to all 96 test tubes to make certain the amount of water added to each test tube is the same irrespective of its position in the standard 96 test tube array.
The configuration and arrangement of the novel multi-layered cell harvester of the present invention not only provides an even distribution of wash fluid simultaneously to each of the 96 test tubes but is furthermore susceptible to robotic operation by an automatic separation of the top multi-layered block from the bottom multi-layered block coupled with an automatic feeding of new sheets of filter paper and the positioning of the filter paper between the two blocks while a new rack of 96 test tubes is introduced into the cell harvester. The novel multi-layered cell harvester includes a number of unique features which assist in the automatic removal, washing and filtering of samples while reducing the labor intensive procedures of the prior art. The novel multi-layered cell harvester further guards against contamination of the various samples while utilizing small quantities of materials from the standard 96, 48 and other test tube racks while reducing the labor, cost and time previously incident to cell harvesting operations.